Staphylococcus aureus (S. aureus) infections are a major public health problem, responsible for debilitating and life-threatening conditions including osteomyelitis and infectious arthritis. Technologies that enable the detection and localization of S. aureus in animals provide a means to assess the life-cycle dynamics, host interactions and antibiotic susceptibility of these bacteria in their natural environment and thus have great value as tools for scientific discovery and medical diagnostics. Current methods for imaging unmodified, naturally occurring S. aureus in animals are limited by the fact that they produce signal prior to encountering their target and most are non-specific with respect to bacterial species. An ideal molecular imaging probe for S. aureus would produce signal only upon encountering the targeted, unmodified bacteria or material derived from it. Such probes would enable the in vivo dynamic imaging of naturally occurring S. aureus strains with superior target-to-background ratios over existing technologies and would facilitate the clinical diagnosis and treatment evaluation of S. aureus infections in humans. The long-term goal of this line of investigation is to develop a novel class of nucleic acid-based activatable imaging probes for the specific non-invasive imaging of various bacterial species in animals and humans. The objective of this application is to demonstrate the utility of nucleic acid-based activatable imaging probes for the detection of S. aureus in vitro and in mice. The central hypothesis of this proposal is tha oligonucleotide-based nuclease substrates with fluorophore-quencher pairs (fluorophore is unquenched upon nuclease digestion), can be tailored via chemical modification to specifically detect nucleases of S. aureus and can thus serve as specific and sensitive probes for detection of the bacteria themselves. Specific aims are: 1) Generate nuclease-activated probes that can specifically detect micrococcal nuclease (MN) of S. aureus. Preliminary studies provide examples of chemically modified oligonucleotide probes that can differentiate between MN and mammalian serum nucleases. The working hypothesis for this aim is that oligonucleotides with the appropriate chemical modifications will be readily digested by MN, but resistant to both mammalian and various bacterial nucleases. Several distinct bacterial and mammalian nucleases will be included in these in vitro experiments. 2) Demonstrate the detection of focal S. aureus infections in mice with nuclease-activated probes. The working hypothesis for this aim is that nuclease- activated probes with quencher/fluorophore pairs that are susceptible to digestion by MN will enable the non- invasive detection and localization of focal S. aureus infections in mice. In summary, the proposed work is expected to result in the development of a robust activated imaging probe-based approach for the non-invasive detection and localization of S. aureus in animals. This contribution is significant because activated imaging probes have critical advantages over existing technology for this problem, and may prove to be generally useful for research and clinical diagnostic applications involving S. aureus.